Fluorescence

1
Fluorescence

• Fluorescent corals

2
(No Transcript)
3
Jablonski Diagram
4

• Fluorescence
• From v 0 down
• Absorption
• From v 0 up
• So expect the emission and absorption spectra to
  overlap here
• Mostly dont because of changes of energy due
  to solvent interactions

5
(No Transcript)
6
Fluorescence is always at a longer wavelength
than absorption, because of the loss of
vibrational energy This is known as Stokes shift
7
Mirror image rule(Kashas Rule)

• The shape of an emission spectrum of a simple
  molecule is the mirror image of the absorption
  spectrum.
• The vibrational levels have approximately the
  same space in the ground state and the first
  excited level because the shape of the molecule
  does not really change.

8
(No Transcript)
9
(No Transcript)
10
(No Transcript)
11
Excited Singlet state paired electrons but one
is excited

• No splitting of electronic energy levels occurs
  when the molecule is in a magnetic field

• Triplet state unpaired electrons the
• spins of the two electrons are parallel
• Paramagnetic
• Triplet because of spectroscopic
• multiplicity

12
Intersystem Crossing

• When the lowest vibrational state of S1 has the
  same energy as an upper vibrational level of the
  triplet state.
• Lifetime of a triplet state is long there are
  lots of chances for loss of energy in collisions
• We do not see phosphorescence in liquids at room
  temperature

13
What molecules fluoresce?

• Rigid, coplanar (reduces collisional dectivation)
• Conjugated
• Have to freeze to get phosphorescence or put on
  filter paper
• Use micelles, cyclodextrin
• All reduce collisional deactivation

14
Note the difference in rigidity
15
Applications
16
Advantages of Fluorescence over Absorption

• Greater selectivity and freedom from spectral
  interferences
• Fewer species which luminesce
• Can vary the absorption (excitation) and emission
  wavelengths
• Lower LOD than Absorption for same compound
• F is linear with conc over 3-4 orders of magnitude

17
Lower LOD than Absorption for same compound

• Fluorescence is read directly by detector
• Absorption is a ratio
• F is linear with conc over 3-4 orders of
  magnitude(extending to lower conc range)

18
Lysergic acid diethylamide

• 50 µg is active
• Plasma or urine
• Make basic
• Extract with 982
• n-heptaneisoamyl alcohol
• Excitation 335 nm
• Emission 435 nm

19
Phosphorescence

• Radiative relaxation from T1 to G
• Is forbidden so has long lifetime
• 10-6 10 sec
• To make a glow-in-the-dark toy, what you want is
  a phosphor that is energized by normal light and
  that has a very long persistence. Two phosphors
  that have these properties are Zinc Sulfide and
  Strontium Aluminate. Strontium Aluminate is newer
  -- it's what you see in the "super"
  glow-in-the-dark toys. It has a much longer
  persistence than Zinc Sulfide does. The phosphor
  is mixed into a plastic and molded to make most
  glow-in-the-dark stuff.

20
Phosphorescence

• Occurs in solids
• Which may be frozen solvents
• Reduces the number of collisions
• Paramagnetic species increase the likelihood of
  intersystem crossing
• So reduce fluorescence and phosphorescence

21
Shape of Emission spectrum

• Does not change with excitation wavelength
• BUT the intensity changes
• The most fluorescence will occur when a lot of
  light is absorbed
• Can find an excitation ? by running an absorption
  spectrum
• Use this to find ?emmax and then ?exmax

22
Quantum Yield F FF number of fluorescence
quanta emitted divided by number of quanta
absorbed to a singlet excited state F F ratio
of photons emitted to photons absorbed
23
(No Transcript)
24
Quenching
25
Xenon arc lamp Laser
High power is more important than stability A
reference system is added to measure the stability
26
Xenon Arc Lamp

• Unstable
• Some portion of initial light goes to reference
  detector to ratio with F signal to compensate for
  changes in lamp intensity
• Sometimes a fluorescent standard of rhodamine is
  included
• May have to restrict intensity of light to
  minimize sample decomposition (photobleaching)

27

• Sources of UV produce ozone. Fan disperses this
  and cools lamp.
• Ozone is toxic but also absorbs certain
  wavelengths
• Detector at right angles to lamp
• Two wavelength selectors
• Slits narrow for high resolution
• Wider(5-10 nm) to give greater sensitivity

28
(No Transcript)
29
L kP0 P P P0 10-abc (Beers law)
kP0 P0 10-abc kP01 10-abc Note L
is proportional to P0
30

• 10-abc e-(2.3abc)
• Expand in a series

31
Calibration using Raman peak for water
Raman peak maxima of water at various Exc ?s Raman peak maxima of water at various Exc ?s
Excitation wavelength/nm Raman emission/nm
200 212
250 272
300 337
350 397
400 463
450 530
500 602
32
Positions of the Raman bands of various solvents when excited at selected wavelengths Positions of the Raman bands of various solvents when excited at selected wavelengths Positions of the Raman bands of various solvents when excited at selected wavelengths Positions of the Raman bands of various solvents when excited at selected wavelengths Positions of the Raman bands of various solvents when excited at selected wavelengths
Solvents Excitation wavelength/nm 313 366 405 436 Excitation wavelength/nm 313 366 405 436 Excitation wavelength/nm 313 366 405 436 Excitation wavelength/nm 313 366 405 436
Water 350 418 469 511
Acetonitrile 340 406 457 504
Cyclohexane 344 409 458 499
Chloroform 346 411 461 502
33
Excitation wavelengths for quinine
Ex 350
Ex 250
450
450
Excitation (Em at 450 nm)
Absorbance
34
(No Transcript)
35
0.05 M H2SO4 Ex at 250 nm
36
Excitation spectrum with emission at 450 nm